JP4956816B2 - Anti-tyrosine kinase antibody and use thereof - Google Patents

Description

  The present invention relates to an antibody against tyrosine kinase, and more particularly to an antibody capable of comprehensively detecting tyrosine kinase without being limited to a specific range.

  When a stimulus from the outside is applied, the cell generates an output corresponding to the input external information. In multicellular organisms, information is input and output between cells. As a result, the life activity of an organism composed of many cells (particularly higher organisms) is maintained. Thus, a cell is a basic structure of a living organism, and various life phenomena are controlled by information transmission between and within the cell.

  There are various methods for transmitting information in a living body, but one of those that play an important role is a pathway through a protein phosphorylation reaction. The protein phosphorylation reaction is a reversible reaction in which a phosphate group of ATP is added to a protein as a substrate by a protein phosphorylase (ie, protein kinase). When phosphorylated, the protein changes its conformation, and as a result, its function is controlled positively or negatively.

  It is estimated that more than 30% of all proteins can be phosphorylated. The number of protein kinases present in eukaryotes is considered to be 1.5% to 2.5% of the total gene product, and is classified into about 200 subfamilies.

Protein kinases can be classified into three types, serine / threonine (Ser / Thr) kinase, tyrosine (Tyr) kinase, and dual specificity kinase, depending on the target amino acid to which the phosphate group is transferred. Serine / threonine kinase is an enzyme that adds a phosphate group to the hydroxyl group of a serine residue or threonine residue of a protein as a substrate. For example, protein kinase A (PKA), protein kinase C (PKC), Ca ²⁺ / Examples include calmodulin-dependent protein kinase (CaMKI, CaMKII, CaMKIV), MAP kinase, and the like. Tyrosine kinase is an enzyme that adds a phosphate group to the hydroxyl group of a tyrosine residue, and Dual specificity kinase (for example, MAP kinase kinase) has functions of both serine / threonine kinase and tyrosine kinase.

  It is known that the protein phosphorylation by these protein kinases is deeply involved in important life phenomena such as metabolic regulation, cell division, differentiation induction, stress response, neurotransmission, and apoptosis. Among others, tyrosine kinases are known to be deeply involved in cell or living body development, differentiation, survival and / or disease.

  The majority of protein kinases are serine / threonine kinases and tyrosine kinases are few. Comparing the two from an evolutionary perspective, serine / threonine kinases are known to be involved in various life phenomena of various organisms ranging from unicellular organisms to multicellular organisms. It has not been seen and has not been reported to plants. Therefore, there is no concept that the knowledge obtained from serine / threonine kinases can be applied to tyrosine kinases as they are, and research is being conducted independently.

  As a result of the human genome project, it was found that 90 types of tyrosine kinase genes exist in humans. Human tyrosine kinases are composed of 32 types of cytoplasmic tyrosine kinases classified into 10 subfamilies and 58 types of membrane-bound tyrosine kinases classified into 20 subfamilies (see Non-Patent Document 1). ).

  Tyrosine kinases were originally found in tumor viruses but were also found to be present in normal organisms. Membrane-bound (receptor) tyrosine kinases are known to transmit extracellular stimuli into cells, function as receptors for various growth factors, and participate in cell morphological changes, proliferation, mobility, etc. ing. Cytoplasmic (non-receptor) tyrosine kinases are known that are involved in the differentiation and differentiation of hematopoietic cells and / or angiogenesis, and the immune mechanism by viable differentiation of T cells and B cells. Many proto-oncogene products have tyrosine kinase activity, and tyrosine kinases are known to regulate cancer growth, metastasis, and / or invasiveness.

  In this way, by analyzing tyrosine kinases that are deeply involved in life phenomena, it is possible to contribute not only to basic life phenomena but also to elucidation of causes, diagnosis and / or treatment of various diseases. Conceivable.

  Conventionally, research on tyrosine kinases has been performed using an antibody (that is, an anti-phosphotyrosine antibody) that specifically recognizes a protein in which a tyrosine residue is phosphorylated (that is, a tyrosine phosphorylated protein). If an anti-phosphotyrosine antibody is used, it can be known whether the tyrosine phosphorylation reaction has arisen in the object which should be investigated.

An anti-phosphotyrosine antibody is an excellent antibody for detecting the degree of tyrosine phosphorylation, but it cannot know the protein expression level of the phosphorylated substrate protein. In this case, the protein expression level of the substrate protein can be estimated by using an antibody against the protein considered to be the substrate protein.
Robinson, DR. , Wu YM. Lin, SF. Oncogene 19, 5548-5557 (2000) Hanks, S .; K. And Hunter, T .; FASEB J.H. 9, 576-596 (1995)

  If a tyrosine phosphorylation reaction has occurred in the subject to be investigated, it is necessary to investigate which tyrosine kinase is responsible for this reaction.

  As antibodies against tyrosine kinases (ie, anti-tyrosine kinase antibodies), only antibodies capable of specifically detecting only a specific protein kinase molecule or a specific protein kinase family have existed so far. Therefore, in order to detect the tyrosine kinase expressed in the subject, there is no means other than using all anti-tyrosine kinase antibodies, and a great deal of labor and cost are required. Furthermore, since the existing anti-tyrosine kinase antibodies have different binding specificities for antigens, the expression levels in the same sample cannot be accurately compared.

  In this way, tyrosine kinases are still expressed in what stage of development, in what tissue, in what situation, how much they are working, how tyrosine kinases are involved. There are many issues that have not been elucidated.

  The present invention has been made in view of the above problems, and its purpose is to establish a method for comprehensively and simply detecting the expression levels of a wide variety of tyrosine kinases present in the living body, The goal is to further develop research on tyrosine kinases.

  The catalytic domain of protein kinase (kinase domain) has 12 common subdomains in terms of primary structure, and the amino acid sequence of that region is highly conserved among protein kinase families (see Non-Patent Document 2). ) Among these twelve subdomains, in particular, subdomain VIB is highly conserved among protein kinases across serine / threonine (Ser / Thr) kinase, tyrosine (Tyr) kinase, dual specificity kinase.

  The present inventors have discovered a tyrosine kinase-specific amino acid sequence in the subdomain VIB sequence present in the catalytic domain of tyrosine kinase, and comprehensively used a number of tyrosine kinases by using peptides generated based on the sequence as antigens. For the first time, an antibody that can be easily detected was obtained, and the present invention was completed.

That is, in order to solve the above-mentioned problem, the peptide according to the present invention is a peptide consisting of the amino acid sequence X ₁ VHRDLX ₂ AX ₃ NX ₄ LV, or a fragment thereof, and includes the amino acid sequence X ₁ VHRDLX ₂ AX ₃ N Where X ₁ is Y or L, X ₂ is R or A, X ₃ is A or R, and X ₄ is I or V.

  The peptides according to the present invention are Val-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn, Val-His-Arg-Asp-Leu-Ala-Ala-Arg-Asn, Tyr-Val-His-Arg. -Asp-Leu-Arg-Ala-Ala-Asn, Leu-Val-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn-Val-Leu, Leu-Val-His-Arg-Asp-Leu-Ala -Ala-Arg-Asn-Val-Leu, Leu-Val-His-Arg-Asp-Leu-Arg-Ala-Arg-Asn-Val-Leu, Leu-Val-His-Arg-Asp-Leu-Ala-Ala -Ala-Asn-Val-Leu or Tyr-Val-His-Arg It is characterized by comprising the amino acid sequence of any of Asp-Leu-Arg-Ala-Ala-Asn-Ile-Leu-Val.

  That is, the method for producing an antibody according to the present invention is characterized by including a step of eliciting an antibody using the peptide as an antigen in order to solve the above problems.

  The method for producing an antibody according to the present invention includes a step of inducing an antibody using a complex of a peptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 3 to 8 and an adjuvant as an antigen. .

  That is, the antibody according to the present invention is preferably produced by the above-described antibody production method.

  The antibody according to the present invention is characterized by specifically binding to a polypeptide having an amino acid sequence represented by any of SEQ ID NOs: 22 to 31.

  The antibody according to the present invention is preferably an anti-tyrosine kinase antibody.

  The antibody according to the present invention is preferably a monoclonal antibody or a polyclonal antibody.

  The immunoassay kit according to the present invention comprises the above-described antibody.

  The immunoassay method according to the present invention is characterized by using the above-mentioned antibody.

  If the antibody according to the present invention is used, the protein expression level of tyrosine kinase can be obtained. Moreover, by using the antibody according to the present invention, it is possible to compare the difference in protein expression level of tyrosine kinase between a normal sample and a disease sample, thereby performing the above comparison in various disease samples, An expression profile can be generated that indicates which tyrosine kinase expression level of which molecular weight is changing in which disease.

(A) Antigen peptide The present inventors have found that a peptide having a specific sequence elicits an antibody capable of comprehensively detecting a wide variety of tyrosine kinases, and has completed the present invention. It should be noted that the term “tyrosine kinase” is intended to mean any enzyme having an activity to phosphorylate tyrosine residues of proteins, unless otherwise mentioned herein.

In one aspect, the present invention provides a peptide comprising the amino acid sequence X ₁ VHRDLX ₂ AX ₃ NX ₄ LV. Preferably, the peptide according to the present invention is a fragment of a peptide consisting of the amino acid sequence X ₁ VHRDLX ₂ AX ₃ NX ₄ LV and comprises the amino acid sequence X ₁ VHRDLX ₂ AX ₃ N. Here, X ₁ is Y or L, X ₂ is R or A, X ₃ is A or R, and X ₄ is I or V.

  As used herein, the term “peptide” is used interchangeably with “polypeptide” or “protein”. A “fragment” of a peptide is intended to be a partial fragment of the peptide. The peptides according to the invention may also be chemically synthesized or isolated from natural sources.

  Synthetic peptides can be synthesized using known methods of chemical synthesis. For example, Houghten is prepared for the synthesis of multiple peptides such as 10-20 mg of 248 different 13 residue peptides that represent a single amino acid variant of the HA1 polypeptide segment prepared and characterized in less than 4 weeks. A simple method is described. Houghten, R.A. A. , Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in US Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure, the individual resins used for solid phase synthesis of various peptides are contained in separate solvent permeable packets, allowing optimal use of many identical iteration steps associated with solid phase methods. Complete manual procedures allow 500-1000 or more syntheses to be performed simultaneously (Houghten et al., Supra, 5134). These documents are incorporated herein by reference.

  The term “isolated” peptide or protein is intended to be a peptide or protein that has been removed from its natural environment. For example, recombinantly produced peptides and proteins expressed in host cells are isolated, as are natural or recombinant peptides and proteins that have been substantially purified by any suitable technique. Conceivable.

  Peptides according to the present invention are naturally purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). Including products produced by recombinant technology.

  The peptide according to the present invention may be in a state in which a polynucleotide according to the present invention described later (a gene encoding the peptide according to the present invention) is introduced into a host cell and the polypeptide is expressed in the cell. Alternatively, it may be isolated and purified from cells, tissues and the like.

  The peptide according to the present invention may contain an additional peptide. Examples of the additional peptide include epitope-labeled peptides such as His, Myc, and Flag. In a preferred embodiment, the peptides according to the invention can be expressed recombinantly in a modified form such as a fusion protein. For example, additional amino acids, particularly charged amino acid regions, of the peptides according to the invention may be used in order to improve stability and persistence in the host cell during purification or subsequent manipulation and storage. Can be added to the N-terminus.

  In one embodiment, the peptide according to the present invention is Val-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn, Val-His-Arg-Asp-Leu-Ala-Ala-Arg-Asn, Tyr- Val-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn, Leu-Val-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn-Val-Leu, Leu-Val-His-Arg- Asp-Leu-Ala-Ala-Arg-Asn-Val-Leu, Leu-Val-His-Arg-Asp-Leu-Arg-Ala-Arg-Asn-Val-Leu, Leu-Val-His-Arg-Asp- Leu-Ala-Ala-Ala-Asn-Val-Leu or Tyr-V It may consist of any of the amino acid sequences of l-His-Arg-Asp-Leu-Arg-Ala-Ala-Asn-Ile-Leu-Val.

  The peptides according to the present invention are very useful as peptide antigens that raise antibodies capable of detecting a wide variety of tyrosine kinases. That is, the peptides according to the present invention are useful in methods and kits for generating antibodies effective for immunoassays. As used herein, the term “immunoassay” intends an assay performed utilizing an immunological binding reaction based on an antigen-antibody reaction. Examples of assays using immunological binding reactions include antibody assays such as Western blot, immunoprecipitation, sandwich ELISA assay, radioimmunoassay, and immunodiffusion assay, and affinity chromatography. These assays use molecules such as avidin and biotin for molecular immobilization and detection. Techniques for preparing these reagents and methods of using them are known to those skilled in the art. In addition, many binding assays are enhanced in detection using labels with pigments, enzymes, radioactive materials or fluorescent materials.

  Those skilled in the art who have read this specification will readily understand that antibody production methods and kits comprising the step of raising an antibody using the peptide as an antigen are also included within the scope of the present invention. In the antibody production method according to the present invention, a complex of the above peptide with an adjuvant may be used as an antigen.

  That is, an object of the present invention is to provide an antigenic peptide capable of generating an antibody capable of comprehensively detecting a wide variety of tyrosine kinases. The peptide (specifically described in the present specification ( Polypeptide) does not exist in the production method or the like. Therefore, it should be noted that peptides capable of generating antibodies capable of comprehensively detecting tyrosine kinases obtained by methods other than the above methods also belong to the scope of the present invention.

(B) Polynucleotide Encoding Antigen Peptide The present invention provides a polynucleotide encoding the peptide according to the present invention. As used herein, the term “polynucleotide” is used interchangeably with “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown.

  The polynucleotide according to the present invention may be produced as a cleaved fragment of a longer polynucleotide (for example, a polynucleotide comprising a full-length cDNA encoding a tyrosine kinase protein) or may be chemically synthesized. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer and the like.

  Examples of the method for obtaining the polynucleotide according to the present invention include a method using an amplification means such as PCR. For example, in the polynucleotide cDNA of the present invention, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and genomic DNA (or cDNA) or the like is used as a template using these primers. By performing PCR or the like and amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained.

  Further, the polynucleotide according to the present invention can be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.

  That is, an object of the present invention is to provide a polynucleotide encoding an antigenic peptide capable of raising an antibody capable of detecting a wide variety of tyrosine kinases, and is specifically described in the present specification. It does not exist in a method for producing a polynucleotide. Therefore, it should be noted that the polynucleotide encoding the peptide obtained by other than the above methods also belongs to the scope of the present invention.

(C) Antibody The present invention provides an antibody capable of detecting a wide variety of tyrosine kinases. The antibody according to the present invention is preferably produced by the above-described antibody production method. The antibody according to the present invention can specifically bind to a peptide having an amino acid sequence represented by any of SEQ ID NOs: 22 to 31, and may be a monoclonal antibody or a polyclonal antibody. The antibody according to the present invention is preferably raised using the above-mentioned peptide as an antigen, but it is also preferable to mix these peptides and use them as an antigen.

  As conventional anti-tyrosine kinase antibodies, many antibodies that can specifically bind to a part or all of a specific family are known. However, such antibodies could not comprehensively detect various tyrosine kinases (90 types in humans) across multiple families.

As used herein, the term “antibody” refers to immunoglobulins (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) ₂ fragments, Fc fragments), eg Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, and humanized antibodies. The antibodies according to the present invention may be useful for selecting biological materials that express tyrosine kinases.

  “Antibodies” can be prepared by various known methods (for example, HarLow et al., “Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, New York (1988)”, Iwasaki et al., “Monoclonal antibody hybridomas and ELISA, Kodansha (91) ").

  Peptide antibodies can be made by methods well known in the art. For example, Chow, M .; Et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, F .; J. et al. Et al. Gen. Virol. 66: 2347-2354 (1985) (incorporated herein by reference). In general, animals can be immunized with free peptide; however, anti-peptide antibody titers can be boosted by coupling the peptide to a macromolecular carrier (eg, keyhole limpet hemocyanin (KLH) or tetanus toxoid). obtain. For example, peptides containing cysteine can be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides can be coupled more like glutaraldehyde. Common linking agents can be used to couple to the carrier. Animals such as rabbits, rats, and mice can be injected either intraperitoneally and / or intradermally with either free or carrier-coupled peptides, eg, emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant. Immunized. To provide useful titers of anti-peptide antibodies that can be detected by an ELISA assay using, for example, free peptide adsorbed on a solid surface, for example, at intervals of about 2 weeks May be needed. The titer of anti-peptide antibodies in serum from immunized animals is increased by selection of anti-peptide antibodies, for example, adsorption to peptides on solid supports and elution of selected antibodies by methods well known in the art. obtain.

As used herein, the term “an antibody that specifically binds to a tyrosine kinase” refers to a complete antibody molecule and antibody fragment (eg, Fab and F (ab ′)) that can specifically bind to a tyrosine kinase. ₂ fragments). Fab and F (ab ′) ₂ fragments lack the Fc portion of the complete antibody, are removed more rapidly by circulation, and have little to no nonspecific tissue binding of the complete antibody (Wahl et al., J Nucl.Med.24: 316-325 (1983) (incorporated herein by reference). These fragments are therefore preferred.

  Furthermore, additional antibodies that can bind to the antigenic peptide can be produced in a two-step procedure through the use of anti-idiotype antibodies. Such a method uses the fact that the antibody itself is an antigen, and thus it is possible to obtain an antibody that binds to a secondary antibody. According to this method, an antibody that specifically binds to a tyrosine kinase is used to immunize an animal (preferably a mouse). The splenocytes of such animals are then used to produce hybridoma cells, and the hybridoma cells produce a clone that produces an antibody whose ability to bind to an antibody that specifically binds tyrosine kinases can be blocked by an antigenic peptide. To be screened. Such antibodies include anti-idiotypic antibodies against antibodies that specifically bind to tyrosine kinases and can be used to immunize animals to induce the formation of antibodies that specifically bind to additional tyrosine kinases. .

It is clear that Fab and F (ab ′) ₂ and other fragments of the antibodies according to the invention can be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (resulting in Fab fragments) or pepsin (resulting in F (ab ′) ₂ fragments). Alternatively, tyrosine kinase binding fragments can be produced by the application of recombinant DNA technology or by synthetic chemistry.

  As described above, phosphorylase (protein kinase) is common to three types of serine / threonine (Ser / Thr) kinase, tyrosine (Tyr) kinase, and dual specificity kinase in that it adds a phosphate to a substrate protein. is doing. Thus, regions that provide features common to all kinases in terms of structure or function (for example, ATP binding sites) are highly conserved throughout the kinase.

  Usually, when an antibody is produced using a region highly conserved in a specific protein family as an antigen, an antibody capable of recognizing the whole family is raised. Therefore, when an antibody is obtained based on the sequence of a specific region (for example, VIB subdomain), an antibody that recognizes serine / threonine (Ser / Thr) kinase or tyrosine (Tyr) kinase separately can be obtained. It is difficult to predict this, and an antibody that can actually distinguish the two based on the sequence of a specific region has never been obtained.

  When the antibody according to the present invention is used, it is useful as a research material for analyzing a signal transduction pathway mediated by tyrosine phosphorylation and its regulatory mechanism, and through such research, various diseases related to the signal transduction system and its regulatory mechanism. Can be effectively used for the pathological analysis of (for example, screening of molecules related to the signal transduction pathway and examination of the correlation between the disease and the signal transduction pathway).

  In addition, by performing affinity purification using the antibody according to the present invention, and subsequently performing amino acid analysis, mass spectrometry, etc., it is possible to identify which tyrosine kinase is a known protein or to find a novel tyrosine kinase. Can do. In addition, the antibody according to the present invention is very useful when conducting research focusing on the whole image of tyrosine kinase, such as expression and kinetic analysis of tyrosine kinase using various materials such as biological tissues, cells, and intracellular organs. Can be a useful research tool.

Thus, it can be said that the antibody according to the present invention only needs to comprise at least an antibody fragment that recognizes the antigenic peptide according to the present invention (for example, Fab and F (ab ′) ₂ fragment). That is, it should be noted that an immunoglobulin comprising an antibody fragment that recognizes the antigenic peptide according to the present invention and an Fc fragment of a different antibody molecule is also included in the present invention.

  That is, an object of the present invention is to provide an antibody capable of comprehensively detecting a wide variety of tyrosine kinases, and includes the individual immunoglobulin types (IgA) specifically described in the present specification. , IgD, IgE, IgG, or IgM), chimeric antibody production methods, antigen peptide production methods, and the like. Therefore, it should be noted that antibodies obtained by methods other than the above methods also belong to the scope of the present invention.

[2] Kit and Method for Detecting Tyrosine Kinase The present invention provides an immunoassay kit for detecting tyrosine kinase. The immunoassay kit according to the present invention is characterized by comprising an antibody that specifically binds to tyrosine kinase in order to detect tyrosine kinase. Preferably, the antibody may be an antibody according to the present invention. The immunoassay kit according to the present invention may include other components known in the art used in general immunoassays in addition to the above-described antibodies.

  The present invention also provides an immunoassay method for detecting tyrosine kinases. The immunoassay method according to the present invention includes a step of incubating with a sample an antibody that specifically binds to tyrosine kinase in order to detect tyrosine kinase. Preferably, the antibody may be an antibody according to the present invention. The immunoassay method according to the present invention may include other steps known in the art used for general immunoassay in the step of administering the antibody.

  By using the immunoassay kit and immunoassay method according to the present invention, by detecting tyrosine kinase in a sample obtained from a normal biological material or a biological material in a pathological state, and comparing the data of both, Expression profiles for tyrosine kinases that specifically alter expression levels in specific diseases can be generated. In addition, those skilled in the art easily understand that the immunoassay kit and the immunoassay method according to the present invention can be used as a diagnostic kit and a diagnostic method.

  As used herein, the term “biological material” intends a source for obtaining a biological sample (a tissue sample or a cell sample obtained from an organism). Biological samples include cell lines, tissue cultures, body fluids (eg, blood, saliva, plaque, serum, plasma, urine, synovial fluid, and cerebrospinal fluid) or other containing protein or DNA or mRNA thereof. Any sample obtained from a source is contemplated. As used herein, the term “sample” includes, in addition to the biological sample and the tissue sample, a genomic DNA sample and / or a total RNA sample extracted from the biological sample and the tissue sample. Also mentioned. If the biological sample contains mRNA, tissue biopsy is a preferred source. Methods for obtaining tissue biopsies and body fluids from mammals are also well known in the art.

  As described above, since the term “immunoassay” includes affinity chromatography, the immunoassay kit according to the present invention can be a kit for affinity purification of tyrosine kinase. The assay method can also be a method of affinity purifying tyrosine kinases.

  The invention is illustrated in more detail by the following examples, but should not be limited thereto.

<I: Procedure>
[1: Materials and methods]
(1-1) Animals Mice (Balb / c, female 5 weeks old) and rats (Wister, male) were purchased from Japan SLC. These animals are bred in a breeding room set at an environmental condition of a temperature of 25 ± 2 ° C., a humidity of 50 ± 20%, and a light / dark cycle of 12 hours in a state where food and water can be freely ingested, and to the breeding room Immunization was carried out after more than one week from the day of admission.

(1-2) Cell Mouse myeloma cell Sp2 / 0 strain (BALB / c mouse-derived myeloma cell, 8-azaguanine resistant, hypoxanthine guanine phosphoryltransferase (HGPRT) -deficient strain), Professor Katsuichiro Okazaki (Faculty of Agriculture, Kagawa University) Room). When used for cell fusion, cells selected in an azaguanine-containing medium in consideration of changes in traits were used.

(1-3) Reagents 50% polyethylene glycol solution (PEG-1500), HAT concentrate (50 ×: 680.5 mg / l hypoxanthine, 8.8 mg / l aminoterine, 193.8 mg / l thymidine) were purchased from GIBCO . Keyhole limpet hemocyanin (KLH) was purchased from Calbiochem. Bovine serum albumin (BSA) and poly-L-lysine (PLL) were purchased from Sigma. Anti-mouse IgG + IgA + IgM goat antibody labeled with horseradish peroxidase (HRPO) was purchased from ICN Pharmaceutical. Anti-His-Tag antibody was purchased from Invitrogen. SuperSignal West Dura Extended Duration Substrate (SuperSignal) was purchased from Pierce. pET-23a (+) and BL21 (DE3) were purchased from Novagen. pGEM-T Easy was purchased from Promega. Each restriction enzyme was purchased from Nippon Gene. Each primer was synthesized at Hokkaido System Science. Rat Ca2 ⁺ / calmodulin-dependent protein kinase II 30 kDa fragment (30K-CaMKII), rat Ca2 ⁺ / calmodulin-dependent protein kinase IV (CaMKKVI), mouse Ca2 ⁺ / calmodulin-dependent protein kinase (CaMKK), rat Ca2 ⁺ / Calmodulin-dependent protein kinase phosphatase (CaMKP) and rat protein phosphatase 2C (PP2C), Tomoko Kinashi, Kagawa University Faculty of Agriculture, Department of Biofunctional Sciences, 2002 graduation thesis, Toshiyuki Tsuge, Department of Bioresource Sciences, Graduate School of Agriculture, Kagawa University It was prepared according to the method described in the 14th master's thesis. cAMP-dependent protein kinase (PKA) was purified from bovine heart muscle. For reagents not specifically described, Wako Pure Chemical Industries or Nacalai Tesque special grade reagents were used.

(4) Peptide synthesis Peptides for use in immunization were synthesized at AIST. In all of these peptides, cysteine was introduced at the N-terminus in order to enable crosslinking with a carrier using a bivalent crosslinking reagent. The synthesized peptides were named 10RAAN, 10AARN, 11RAAN, 13RAAN, 13AARN, 13RARN, 13AAAN, and 14RAAN (FIG. 1B).

[2: Preparation of polyclonal antibody]
(2-1) Preparation of KLH-peptide complex 2 mg of KLH as a carrier protein was dissolved in 0.2 ml of 10 mM phosphate buffer (pH 7.2), and 25 mg / ml N- (6-maleimidocaproxy) succinimide (EMCS) 22 μl was added and allowed to react at room temperature for 30 minutes while stirring with a stirrer. The reaction solution was centrifuged at 15,000 rpm for 5 minutes at room temperature, and the supernatant was separated on a Sephadex G-50 Fine (Amersham Biosciences) column pre-equilibrated with 50 mM phosphate buffer (pH 6.0). Gel filtered. For the eluted fraction from the column, the absorbance at 280 nm was measured, and the carrier protein peak was recovered. 0.2 ml of a 10 mg / ml antigen peptide solution was added to the recovered carrier protein fraction, and the mixture was incubated for 2 hours or more with stirring. The pH of the reaction solution was measured using a pH test paper, and the reaction solution was adjusted to pH 7 or higher using 0.4 M Na ₂ HPO ₄ and further incubated for 3 hours or more. The same operation was repeated, and when the drop in pH could not be confirmed, dithiothreitol (DTT) was added to a final concentration of 5 mM and the reaction was stopped by stirring for 1 hour to obtain a peptide complex. The prepared peptide complex was stored at −30 ° C. until use.

(2-2) Preparation of emulsion and immunization to mice A KLH-peptide complex (corresponding to 100 μg of peptide per mouse) was converted into phosphate buffered saline (PBS) (2.68 mM KCl, 1.47 mM KH ₂ PO _4. , 137 mM NaCl, 8.1 mM Na ₂ HPO ₄ ) to 0.2 ml, mixed with the same amount of complete Freund's adjuvant (FCA; DIFCO Laboratories) in a 2.5 ml syringe, and then sonicated. To make an emulsion.

  Immunize the mouse by injecting 0.4 ml (100 μg) of the resulting emulsion subcutaneously with a 25G needle on the back and ventral side of the front and back of the ether-anesthetized mouse. did. Two weeks after immunization, an emulsion was prepared using incomplete Freund's adjuvant (DIFCO Laboratories) instead of Freund's complete adjuvant, and a second immunization was performed as in the first immunization. Thereafter, every two weeks, mice were injected intraperitoneally with KLH-peptide complex (equivalent to 50 μg of peptide per mouse) diluted to 100 μl with PBS. Three or more mice were used for one type of antigen, and all were immunized under the same conditions.

(2-3) Preparation of mouse antiserum After the second immunization, partial blood collection was performed by eye blood collection of the mouse approximately 1 week after each immunization, and from the carotid artery and heart of the ether anesthetized mouse approximately 1 week after the final immunization. A whole blood collection was performed. The collected blood was allowed to stand at room temperature for 1 hour and then allowed to stand overnight at 4 ° C. The blood was centrifuged at 15,000 rpm for 15 minutes at 2 ° C., and the supernatant was collected as antiserum and stored at −80 ° C. until use.

[3: Preparation of monoclonal antibody]
(3-1) Cell fusion and hybridoma culture Mice were immunized with peptides (13 RAAN and 11 RAAN), and BALB / c mice with an increased antibody titer were selected. The mice were anesthetized with ether 4 days after the final immunization, disinfected with 70% ethanol from neck to abdomen, and whole blood was collected from the carotid artery and heart. Thereafter, the abdomen was opened and the spleen was removed. The excised spleen was immersed in PBS in a petri dish to remove adipose tissue and connective tissue, and then washed 5 times with 2 petri dishes (10 times in total). After moving the spleen in the petri dish into the safety cabinet, the spleen was washed in the same manner, cut into the center of the spleen, and transferred to a Dounce homogenizer. To this homogenizer, 5 ml of Dulbecco's modified Eagle's medium (DMEM, Sigma) (DMEM (−)) not containing fetal bovine serum (FBS) was added, and the tissue was gently disrupted. Transfer to tube. After adding 5 ml of DMEM (−) to the homogenizer and lightly pipetting, it was transferred to the same tube and centrifuged at 1,000 rpm at room temperature for 5 minutes. After removing the supernatant, ₃ ml of erythrocyte lysis solution (829 mg NH ₄ Cl, 100 mg KHCO ₃ , 37 mg / 100 ml EDTA 2Na (pH 7.4)) was added to the cell pellet, and the erythrocytes were destroyed by light pipetting. This solution was mixed with 10 ml of DMEM (−) and centrifuged at 1,000 rpm at room temperature for 5 minutes. After removing the supernatant, 10 ml of DMEM (−) was added to the cell pellet, and lightly pipetted. This solution was centrifuged at 1,000 rpm at room temperature for 5 minutes, and then the supernatant was removed to remove cells. Was washed. After performing this washing operation three times, the cells were suspended in 10 ml of DMEM (−) to obtain a spleen cell suspension.

  The cultured myeloma cells were suspended by pipetting and collected by centrifugation, then washed three times with 10 ml of DMEM (−) (centrifuged at 1,000 rpm at room temperature for 5 minutes), and in 10 ml of DMEM (−). To obtain a myeloma cell suspension.

  A part of the cell suspension was diluted 10-fold with DMEM (−). Spleen cells were further mixed with an equal volume of trypan blue solution (0.2% trypan blue, 0.8% sodium chloride) to stain dead cells, and then a Burker-Turk hemocytometer was used. Used to count the number of viable cells.

For 13 RAAN immunized mice, cell fusion was performed with 1 myeloma cell for 7 spleen cells, and for 11 RAAN immunized mouse with 1 myeloma cell for 5 spleen cells. Mix the total amount of spleen cells (about 1.2 × 10 ⁸ cells) and myeloma cells (about 1.7 × 10 ⁷ cells for 13 RAAN, about 2.4 × 10 ⁷ cells for 11 RAAN) in a 50 ml tube, After centrifugation at 1,000 rpm for 5 minutes at room temperature, the supernatant was completely removed. The cells were loosened while tapping the tube, and the spleen cells and myeloma cells were mixed well. The cells were fused by adding 1 ml of a 50% polyethylene glycol solution drop by drop with a syringe over 1 minute while tilting and rotating the tube. Similarly, 7 ml of DMEM (−) was added to this solution over 7 minutes while rotating the tube, and 8 ml of DMEM (10% FBS medium) containing 10% FBS was gently added. The mixed solution was centrifuged at 1,000 rpm at room temperature for 5 minutes, and then the supernatant was removed. To the cell pellet, 2 ml of 10% FBS medium was added, and the cells were loosened while tapping the tube. After gently adding 42 ml of 10% FBS medium to the tube and suspending the cells by inversion, the volume was adjusted to 50 ml with the same medium. The cell suspension was dispensed into four 48-well multiplates (Sumitomo Bakelite) at a rate of 0.25 ml per well, and the cells were cultured at 37 ° C., 5% CO ₂ , and wet conditions. On the next day (within 24 hours), 10% FBS medium containing a double concentration of HAT was added in an amount of 0.25 ml per well. Thereafter, half of the HAT selection medium was changed every 3 days, and selective culture of hybridomas was performed.

(3-2) Screening of positive wells About 10 days after cell fusion, 0.1 ml of the culture supernatant of the hybridoma was collected, and antibody-producing positive wells were collected by a dot immunobinding assay (DIA) described later. Screened.

(3-3) Cloning The wells with positive screening results were cloned by limiting dilution. At this time, the prepared mouse thymocytes were usually used as feeder cells, but as an alternative, Bryclone (Dainippon Pharmaceutical Co., Ltd.), a hybridoma growth auxiliary reagent, was used.

0.1 ml of 10% FBS medium pre-added with 2 × HT and 10% Blyclone was added to each well of a 96-well multiplate (Nunc), and the plate was kept at 37 ° C., 5% CO ₂ until use. And wet conditions (cell culture conditions).

Cells in wells with positive screening results were floated by pipetting, and the number of viable cells was counted using a Burker-Turk type hemocytometer. A cell suspension diluted with 10% FBS medium so that the number of cells per 0.1 ml is 20 is dispensed into each of the above 96-well multiplates in 0.1 ml portions, and these cells are incubated at 37 ° C., 5 ° C. The cells were cultured under% CO ₂ and humid conditions. After culturing for about 1 week, each well was observed to confirm that colonies were formed. Screening by the DIA method was performed, and the cell suspension was diluted with 10% FBS medium so that the number of cells per 0.1 ml was 2 by limiting dilution for antibody production positive wells. This cell suspension was dispensed in 0.1 ml portions into a 96-well multiplate, and then secondary cloning was performed. The same operations as in the secondary cloning were performed on the wells in which the number of colonies was 1 and the antibody production was positive in the secondary cloning. After screening, it was confirmed that all wells were positive for antibody production, and cloning was terminated. About the obtained cell, 6-well plate, 94mm dish (greiner bio-one) and the culture scale were expanded sequentially, and it established as an antibody production hybridoma.

(3-4) Cryopreservation of cells The established hybridoma was cultured in a 94 mm dish and cryopreserved when it became confluent. For cryopreservation, a final concentration of 10% dimethyl sulfide (DMSO) as a cryoprotectant was added to a storage medium (20% FBS). About 5 × 10 ⁶ cells were suspended in 1 ml of this medium and stored at −80 ° C.

(3-5) Preparation of Monoclonal Antibody from Mouse Ascites Six-week-old BALB / c mice were injected intraperitoneally with 0.5 ml of pristane, and about 1 × 10 ⁸ cells of hybridoma was added to 10% FBS medium after about 1 week. The suspension was suspended in 0.5 ml, and this suspension was injected intraperitoneally into mice. The mice were observed for ascites retention after the hybridoma administration. When ascites was sufficiently accumulated, the mice were anesthetized with ether and whole blood was collected from the carotid artery and heart, and then the abdomen was opened to collect ascites. The collected blood was treated in the same manner as when preparing antiserum. Ascites fluid was centrifuged at 5,000 rpm and 2 ° C. for 15 minutes after collection, and the supernatant was collected and stored at −80 ° C. until use.

[4: Dot immunobinding assay (DIA)]
(4-1) Preparation of PLL-peptide complex 20 μl of 10 mg / ml PLL and 10 μl of 100 mM phosphate buffer (pH 7.2) were mixed, adjusted to 0.1 ml with purified water, and then 25 mg / ml EMCS was added. 11 μl was added, and the mixture was reacted at room temperature for 30 minutes while stirring with a stirrer. In order to remove unreacted substances, extraction with dichloromethane was repeated, and the aqueous layer was recovered when the white intermediate layer formed at the interface became invisible. 5 μl of 10 mg / ml antigen peptide solution was added to 25 μl of the collected aqueous layer and adjusted to 50 μl with purified water, and this mixture was reacted at room temperature for 3 hours or more while stirring. The resulting peptide complex was stored at −30 ° C. until use.

(4-2) Dot immunobinding assay (DIA)
A nitrocellulose membrane (Schleicher & Schuell) with squares of 4 mm × 4 mm was immersed in purified water with a ballpoint pen and shaken at room temperature for 5 minutes. After drying at 50 ° C. for 30 minutes, 0.5 μl each of the peptide PLL-complex (20 μg / ml) used as an antigen at the time of immunization was spotted at the center of each square and irradiated with white light for 30 minutes. Antigen peptide was immobilized on the membrane. The membrane on which the antigen peptide was immobilized was immersed in blocking buffer (5% skim milk (DIFCO Laboratories), PBST (PBS containing 0.05% Tween 20)) and shaken at room temperature for 1 hour. The blocked membrane was cut into divided sizes, each of which was transferred to a well of a 96-well multiplate so that the spot surface was on top, and 0.1 ml of the culture supernatant described above was added to each well as a primary antibody. After incubating at room temperature for 2 hours, the culture supernatant in each well was removed, and the wells were washed 3 times with 0.1 ml of PBST for 5 minutes. Further, 0.1 ml of HRPO-labeled goat anti-mouse IgG + IgA + IgM diluted 1000-fold with blocking buffer was added to each well as a secondary antibody, and the mixture was allowed to react at room temperature for 1 hour with shaking. After completion of the reaction, the membrane was washed twice with PBST and twice with PBS, and a 3,3′-diaminobenzidine (DAB) solution (50 mM Tris-HCl (pH 7.5), 0.05% DAB, 0.01% H ₂ O ₂ ) was added to the well to perform a color reaction. Color development was stopped by washing with water. A mouse antiserum collected before cell fusion was diluted 100-fold with a blocking buffer and used as a primary antibody positive control.

[5: Preparation of tissue extract and cell extract]
(5-1) Preparation of rat cerebral extract The cerebrum extracted from the rat was weighed and transferred to a Teflon-glass homogenizer, and the ice-cold extraction buffer A (5 mM Tris- 5 times the weight (g) (ml). HCl (pH 7.5), 0.5 mM EGTA, 1 mM EDTA, 2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF)) was added. In an ice bath, the brain was completely crushed with a homogenizer (800 rpm) and further stroked up and down 12 times to obtain a brain tissue extract. This brain tissue extract was stored frozen at −30 ° C. until use.

(5-2) Preparation of Neuro2a cell extract Mouse neuroblastoma cell Neuro2a was cultured in a 94 mm dish at 37 ° C., 5% CO ₂ under humid conditions in 10% FBS medium. After removing the medium, 0.5 ml SDS sample buffer was added to the dish to lyse the cells, and this lysate collected in a tube was boiled for 5 minutes to obtain a sample for electrophoresis.

[6: Protein quantification]
The protein concentration of the sample was determined based on a BSA concentration standard curve according to a modified Lowry method (Bensadoun and Weinstein method).

[7: SDS-PAGE, CBB staining and Western blotting]
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 10% acrylamide separation gel and a 3% acrylamide concentration gel according to the method of Laemmli. The separated protein was stained by shaking the gel after SDS-PAGE in Coomassie brilliant blue (CBB) solution (0.25% CBB, 50% trichloroacetic acid) for 10 minutes.

  Western blotting was performed according to the following procedure. Proteins separated by SDS-PAGE are electrically transferred to a nitrocellulose membrane in a blotting buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol) (4 ° C., 300 mA, 1 hour), and then the membrane after transfer Was immersed in blocking buffer and shaken at room temperature for 1 hour. The membrane after blocking was incubated with a primary antibody solution diluted 2-200 times with a blocking buffer at room temperature for 2 hours, washed with PBST for 5 minutes × 3 times, and then incubated with a secondary antibody at room temperature for 1 hour. . As a secondary antibody, HRPO-labeled goat anti-mouse IgG + IgA + IgM was used after being diluted 1,000 times with a blocking buffer. Next, after completion of the antibody reaction, the membrane was washed twice with PBST and twice with PBS, and then reacted with SuperSignal at room temperature for 5 minutes. The chemiluminescence of the antibody reaction positive band was detected with an X-ray film (Fuji Photo Film).

[8: Preparation of Src mutant]
(8-1) Construction of Expression Vector by Inverse PCR Method Using pET-src obtained in this laboratory as a template (template), an upstream primer (5′-TTC GCT AGC GGC) to which an NheI site (underlined portion) has been added. PCR using AGC AAC AAG AGC AAG CC-3 ′: SEQ ID NO: 9) and downstream primer (5′-CTC TCG AGT AGG TTC TCC CCG GGC TGG-3 ′: SEQ ID NO: 10) with an XhoI site (underlined) PGEM-T Easy src was constructed by incorporating the fragment amplified in step 1 into the multiple cloning site of pGEM-T Easy.

  srcRN-5 ′ upstream primer (5-CGA AAT ATC CTA GTA GGG GAG AAC CTG G-3 ′: SEQ ID NO: 11) and srcAA-3 ′ downstream primer (5′-GGC TGC AAG GTC CCG GTG CAC ATA GTT-3 ′ : PET-src AARN fragment was obtained by PCR using SEQ ID NO: 12). The pET-src RARN fragment was obtained by PCR using the srcRN-5 'upstream primer and the srcRA-3' downstream primer (5'-GGC TCG AAG GTC CCG GTG-3 ': SEQ ID NO: 13). The pET-src AAAN fragment was obtained by PCR using the srcAN-5 'upstream primer (5'-GCC AAT ATC CTA GTA GGG GAG AAC-3': SEQ ID NO: 14) and the srcAA-3 'downstream primer. srcIH-5 ′ upstream primer (5′-ATC CAC CGG GAC CTT GCA GCC CG-3 ′: SEQ ID NO: 15) and srcNY-3 ′ downstream primer (5′-ATA GTT CAT CCG CTC CAC ATA GGC-3 ′: sequence The pET-src YIHR fragment was obtained by PCR using number 16). srcVH-5 ′ upstream primer (5′-GTG CAC CGG GAC CTT GCA GC-3 ′: SEQ ID NO: 17) and srcNL-3 ′ downstream primer (5′-CAG GTT CAT CCG CTC CAC ATA GGC C-3 ′: sequence The pET-src LVHR fragment was obtained by PCR using number 18). A pET-src FVHR fragment was obtained by PCR using srcVH-5 'upstream primer and srcNF-3' downstream primer (5'-GAA GTT CAT CCG CTC CAC ATA GGC-3 ': SEQ ID NO: 19). The pET-src ATRN fragment was obtained by PCR using the srcRN-5 'upstream primer and the srcAT-3' downstream primer (5'-GGT TGC AAG GTC CCG GTG CAC-3 ': SEQ ID NO: 20). The pET-src FIHR fragment was obtained by PCR using the srcIH-5 'upstream primer and the srcNF-3' downstream primer. The pET-src CIHR fragment was obtained by PCR using the srcIH-5 'upstream primer and the srcNC-3' downstream primer (5'-GCA GTT CAT CCG CTC CAC ATA GGC-3 ': SEQ ID NO: 21). In addition, pET-src YAHR, pET-src LVHR, pET-src FVRHR, pET-src Easy src as a template for pET-src AARN, pET-src RARN, and pET-src AAAAN. FIHR, pET-src For CIHR, pET-src AARN is used as a template, for pET-src ATRN, pET-src FVHR is used as a template, and GeneAmp PCR System 2700 (Applied Biosystem) is used for PyrobestRase DNA I went there. pET-src AARN, pET-src RARN, pET-src AAAN, pET-src YIHR, pET-src LVHR, pET-src FVHR, pET-src FIHR, pET-src CIHR at 98 ° C. for 10 seconds The fragments were amplified by repeating 30 cycles of annealing at 63 ° C. for 10 seconds and extension reaction at 72 ° C. for 7 minutes, and for pET-src ATRN, heat denaturation at 98 ° C. for 10 seconds, 59.8 ° C. for 10 seconds The fragment was amplified by repeating 30 cycles of annealing and extension reaction at 72 ° C. for 7 minutes. Subsequently, for pET-src AARN, pET-src RARN, and pET-src AAAN, the PCR product (fragment) was isolated by agarose gel electrophoresis, digested with NheI and XhoI, and then the multiple cloning site of pET-23a (+) (FIG. 3A).

(8-2) Expression and purification in E. coli Various Src expression vectors and pET-23a (+) were introduced into E. coli BL21 (DE3) (Novagen) by electroporation, and this E. coli was added to 5 ml of LB / ampicillin liquid medium. (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 100 μg / ml ampicillin) was cultured with shaking at 37 ° C. for 12 hours. Centrifugation of 1 ml of the culture solution collects the cells, and the cells are collected in 0.1 ml of a homogenizing buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 40). Was sonicated. The obtained bacterial lysate was centrifuged (15,000 rpm, 4 ° C., 10 minutes), the supernatant was removed, and the cells were again centrifuged and washed with 0.1 ml of homogenizing buffer. To the resulting precipitate, 0.1 ml of a homogenizing buffer was added and suspended to prepare an electrophoresis sample.

SrcRAAN (WT), SrcAARN, SrcRARN, and SrcAAAN were further purified. The expression vector was introduced into E. coli BL21 (DE3) by electroporation, and the bacterium was cultured with shaking at 37 ° C. for 12 hours in 100 ml of LB / ampicillin liquid medium prepared in a 300 ml flask. The culture solution was centrifuged (5,000 rpm, 4 ° C., 10 minutes) to recover the bacterial cells, and the bacterial cells were ultrasonically disrupted in 10 ml of homogenizing buffer. The obtained bacterial crushing solution was centrifuged (5,000 rpm, 4 ° C., 10 minutes) to separate into a soluble fraction and an insoluble fraction. A solubilization solution (6M guanidine hydrochloride, 50 mM Tris-HCl (pH 7.5), 10 mM 2-mercaptoethanol) was added to the insoluble fraction, followed by sonication to completely solubilize the insoluble fraction. The solubilized insoluble fraction was applied to a HiTrap Chelating HP column (1 ml) that had been equilibrated in advance with a solubilization solution. Thereafter, 10 ml of solubilizing solution, 10 ml of solubilizing solution containing 20 mM imidazole, and 10 ml of solubilizing solution containing 50 mM imidazole were passed through the column to remove non-specifically bound proteins. The His ₆ -Src and His ₆ -Src mutants were then eluted with 10 ml of solubilization solution containing 200 mM imidazole. The fraction of the solubilized solution containing 200 mM imidazole was fractionated into 10 1 ml portions. Thereafter, each fraction was subjected to SDS-PAGE to confirm the eluted fractions of Src and Src mutants. Each eluted sample was placed in a dialysis tube, and 2 times x 3 times against 500 ml of homogenizing buffer. Dialysis was performed to remove imidazole and nickel ions and guanidine hydrochloride in the solution. After dialysis, each sample was stored at −30 ° C. until use.

<II. Result>
[1: Preparation of polyclonal antibody and its reaction specificity]
The inventors have determined that the subdomain VIB present in the catalytic domain of tyrosine kinase has an amino acid sequence different from that of other protein kinases, and that the RAAN sequence found only in Src family tyrosine kinases. And AARN sequences found in other tyrosine kinases were found (SEQ ID NOs: 32-45 (FIG. 1A)). Therefore, nine peptides shown in Table 1 were synthesized based on these amino acid sequences (SEQ ID NOs: 1 to 8 (FIG. 1B)), and a mouse was immunized with a complex of these peptides and KLH to produce antibodies. Evoked.

Moreover, in order to investigate the reactivity of the antiserum obtained from the immunized mouse, Src mutants (SrcAARN, SrcRARN, SrcAAAN) were prepared (SEQ ID NOs: 22 to 25 (FIG. 2A)). Specifically, an Src mutant expression vector prepared using an inverse PCR method was introduced into E. coli BL21 (DE3), and the Src mutant was expressed as a fusion protein with a His ₆ tag at the carboxyl terminus. Since the expressed Src mutant was recovered in a large amount in the insoluble fraction of the E. coli extract, the Src mutant was purified to a single band (about 60 kDa) using a nickel affinity column after solubilization.

  FIG. 2B shows the results of examining the reactivity of the obtained antiserum by Western blotting using Src and Src mutants as analysis samples. These antisera have various reactivity depending on the immunized peptide, and it was found that 11RAAN, 13RAAN, 13AARN, and 14RAAN are antibodies capable of recognizing four types of Src. In addition, 11 RAAN peptide was shown to be effective in obtaining antiserum having broad specificity and strong reactivity.

[2: Preparation of monoclonal antibody and its reaction specificity]
For 11 RAAN peptide-immunized mice in which antibodies with broad specificity and strong reactivity were raised, and 13 RAAN peptide-immunized mice in which antibodies with low reactivity but broad specificity were raised, the spleen was removed 4 days after the final immunization. The prepared splenocytes were fused with myeloma cells. Thereafter, HGRPT-positive strains (nucleic acid synthesis salvage pathway-deficient strains) were selected and screened using a HAT selection medium. As a result of screening 192 wells by DIA using a nitrocellulose membrane spotted with an antigenic peptide, there were 7 positive wells from 11 RAAN peptide-immunized mice and 44 from 13 RAAN peptide-immunized mice, although there was a difference in signal intensity. Positive wells were obtained. Thereafter, wells that were particularly reactive among positive wells (2 wells for 11 RAAN and 8 wells for 13 RAAN) were collected and screened after the primary cloning and when the cells formed colonies. As a result, 56 wells in 192 wells were positive in 11 RAAN, and 119 wells in 768 wells were positive in 13 RAAN. Further, 2 wells for 11 RAAN and 5 wells for 13 RAAN were subjected to secondary cloning and then screened. As a result, 31 wells in 116 wells for 11 RAAN and 34 wells in 112 wells for 13 RAAN were positive. In the obtained positive wells, wells consisting of a single colony were subjected to tertiary cloning and then screened. All wells were positive. Therefore, it was judged that cloning was completed.

  For each positive well, two hybridomas were established from 11 RAAN and one from 13 RAAN. Hybridomas derived from 11 RAAN were named YK34 and YK84, and hybridomas derived from 13 RAAN were named YK68.

  We examined how much tyrosine kinases can be recognized by the monoclonal antibodies produced by these hybridomas. Specifically, the amino acid sequence of subdomain VIB of tyrosine kinase existing in humans was examined, and an Src mutant having an amino acid sequence highly conserved among tyrosine kinases was produced.

  The present inventors obtained amino acid sequences of 90 types of tyrosine kinases existing in humans from a database, and compared sequences corresponding to subdomain VIB. As a result, subdomain VIB did not exist in two of 90 types of tyrosine kinases (EphX and AATYK3). Therefore, the present inventors found amino acid variations in the subdomain VIB sequence for the other 88 types, and classified them into 29 types and 7 types (data not shown).

Among the amino acid sequences of subdomain VIB thus classified, Src mutants (SrcYIHR, SrcLVHR, SrcFVHR, SrcATRN, SrcFIHR, SrcCIHR) were prepared based on sequences found in many tyrosine kinases (SEQ ID NOs: 26 to 31). (FIG. 3A)). Specifically, an Src mutant expression vector prepared using an inverse PCR method was introduced into E. coli BL21 (DE3), and the Src mutant was expressed as a fusion protein with a His ₆ tag at the carboxyl terminus.

  Since the expressed Src mutant was recovered in a large amount in the insoluble fraction of the Escherichia coli extract, production of three types of hybridomas (YK34, YK84, and YK68) was performed using the insoluble fraction as an analysis sample. The reactivity of the monoclonal antibodies (14-2, C4, and H4, respectively) was examined by Western blotting. The YK34-derived antibody (C4) reacted strongly with Src and all Src mutants, among them very strongly with Src and SrcRARN (FIG. 3B). FIG. 3C shows the results using an anti-His antibody and shows that wild-type and mutant Src proteins are all expressed equally. 3B and 4C, lane 1: mock, lane 2: SrcRAAN, lane 3: SrcAARN, lane 4: SrcRARN, lane 5: SrcAAAN, lane 6: SrcYIHR, lane 7: SrcLVHR, lane 8: SrcFVHR, lane 9: SrcATRN, Lane 10: SrcFIHR, Lane 11: SrcCIH.

  Although the antibody (14-2) derived from YK68 reacts with Src and all Src mutants, its reactivity is very weak as in the case of the polyclonal antibody. Could not be raised (data not shown). YK84-derived antibody (H4) reacts with Src and all Src mutants, among which Src and SrcRARN react strongly, but the reactivity with SrcYIHR, SrcFIHR and SrcCIHR was slightly weak (data not shown) ).

  Furthermore, the results of examining the reactivity of the monoclonal antibody with Ser / Thr kinase or other proteins by Western blotting are shown in FIG. FIG. 4A shows the result of CBB staining, indicating that protein is present in each lane of the gel. Neither YK34-derived antibody (C4) nor YK84-derived antibody (H4) reacts with Ser / Thr kinases 30K-CaMKII, CaMKIV, CaMKK and PKA, and also reacts with known protein phosphatases CaMKK and PP2C. It was confirmed that they did not (FIGS. 4B and 4C). Next, to examine whether the antibody derived from YK34 (C4) and the antibody derived from YK84 (H4) can detect tyrosine kinases in tissue or cell extracts, these antibodies and rat cerebral extracts or The reactivity with the Neuro2a cell extract was examined. As a result, it was confirmed that these antibodies react with many proteins contained in rat cerebral extract and Neuro2a cell extract (FIGS. 4B and 4C). Most of the reacted proteins were proteins of 50 kDa or more, and a large number of bands with the same or different mobility were detected between the rat cerebral extract and the Neuro2a cell extract (FIGS. 4B and 4C). From these results, it was shown that the obtained monoclonal antibodies C4 and H4 react with various tyrosine kinases among protein kinases. 4A to C, lane 1: SrcRAAN, lane 2: 30K-CaMKII, lane 3: CaMKIV, lane 4: CaMKK, lane 5: PKA, lane 6: CaMKP, lane 7: PP2C, lane 8: rat brain Extract, lane 9: Neuro2a cell extract.

  The present invention is very useful as a research tool for tyrosine kinases. Furthermore, if the present invention is used, it can greatly contribute to elucidation of the cause, development of means for diagnosis or treatment, and pharmaceutical development in various diseases involving tyrosine kinases.

It is a figure which shows the amino acid sequence alignment (A) about the subdomain VIB of various kinases, and the amino acid sequence (B) of the peptide produced in order to raise the antibody which concerns on this invention. FIG. 3 shows amino acid sequence alignment (A) for Src tyrosine kinase and mutant subdomain VIB, and Western blotting results using various antisera or anti-His antibodies (B) for Src tyrosine kinase and mutant. . Amino acid sequence alignment (A) for Src tyrosine kinase and mutant subdomain VIB, Western blotting results using monoclonal antibody (C4) for Src tyrosine kinase and mutant (B), and Western using anti-His antibody It is a figure which shows the result (C) of blotting. The results of Western blotting using a monoclonal antibody (H4) and gel (A) stained with CBB after various SDS-PAGE (A), Western blotting using monoclonal antibody (C4) (C) are shown. FIG.

Claims (9)

Amino acid sequence Cy s -Tyr-Val-His- Arg-Asp-Leu-Arg-Ala-Ala-As n or peptide, characterized in that Ranaru.   A method for producing an antibody comprising the step of raising an antibody using the peptide of claim 1 as an antigen.   A method for producing an antibody comprising the step of raising an antibody using the complex of the peptide of claim 1 and an adjuvant as an antigen.   An antibody produced by the method for producing an antibody according to claim 2 or 3.   The antibody according to claim 4, wherein the antibody specifically binds to a polypeptide having an amino acid sequence represented by any of SEQ ID NOs: 22 to 31.   6. The antibody according to claim 4 or 5, which is an anti-tyrosine kinase antibody.   The antibody according to any one of claims 4 to 6, which is a monoclonal antibody or a polyclonal antibody.   An immunoassay kit comprising the antibody according to any one of claims 4 to 7.   An immunoassay method using the antibody according to any one of claims 4 to 7.

Images